强啡肽在心肌缺血预处理时的变化及对缺血再灌注心肌的保护作用

发布时间：2018-03-20 07:11

  本文选题：强啡肽　切入点：к阿片受体　出处：《第四军医大学》2007年硕士论文　论文类型：学位论文

【摘要】： 阿片肽和阿片受体在体内广泛分布,长期以来人们一直认为阿片肽是一种中枢神经肽或神经递质,仅在中枢神经系统发挥作用。近20年来研究发现心脏也可合成内源性阿片肽,通过自分泌或旁分泌的方式对自身功能进行调节,其作用是通过各自相应的阿片受体所介导的。与心脏功能密切相关的阿片受体主要是μ、δ和к三种亚型,其中在心血管系统占主导地位的阿片受体亚型是к阿片受体,强啡肽(Dyn)是к阿片受体的内源性配体。 研究证实,心肌缺血预处理(IPC)能够使心脏对随后的持续的心肌缺血再灌注(I/R)损伤的耐受性提高,IPC对缺血心肌的这种保护作用主要是由于缺血触发释放内源性物质实现的。已知在正常条件下,心脏可合成Dyn并能激活κ阿片受体产生对心血管系统的调节作用;在心肌缺血时,内源性Dyn有可能释放增多并通过激动κ阿片受体参与心肌缺血的病理过程。因此本试验旨在研究内源性Dyn是否在IPC心肌保护作用中扮演重要的角色以及激活κ阿片受体是否对缺血再灌注心肌具有直接的保护作用。 1、目的: (1)观察在IPC过程中,Dyn在血浆水平的浓度变化以及Dyn在心肌组织mRNA表达的变化。 (2)给以κ阿片受体的选择性激动剂,观察其对大鼠心肌I/R损伤的影响。 (3)给以κ阿片受体的选择性阻断剂,观察其对IPC的心肌保护作用的影响。 2、研究方法: (1) IPC模型制备方法:大鼠冠状动脉左前降支(LAD)距左心耳下缘约2 mm处穿线,套扎LAD,心肌缺血5 min,松开,再灌注5 min,3个循环。 (2) I/R模型制备方法:套扎LAD,心肌缺血45 min,松开,再灌注120 min。 (3)应用生理学整体实验技术进行大鼠血液的采集和心脏功能的测定。 (4)采用RT-PCR方法观察大鼠心肌组织Dyn mRNA表达的变化。 (5)采用放射免疫分析方法测定血浆中Dyn,磷酸肌酸激酶(CK)和乳酸脱氢酶(LDH)的水平。 (6)应用双重染色法检测大鼠心肌梗死面积。 3、主要结果: (1)采集各组大鼠血液,离心取上层血浆,测定各组大鼠血浆中Dyn的浓度,IPC组(缺血5 min,再灌注5 min,3个循环后采样)的浓度显著高于同时间sham组(不给予缺血和再灌注,其余处理同IPC组)(P0.05);IPC组的浓度显著高于con组(未给予任何处理)(P0.05)。 (2)取大鼠心肌组织,观察各组心肌组织Dyn mRNA表达的变化,IPC组(缺血5 min,再灌注5 min,3个循环后采样)强啡肽基因表达水平与同时间sham组(不给予缺血和再灌注,其余处理同IPC组)和con组(未给予任何处理)相比,无显著差别(P0.05)。 (3)静脉给予选择性κ阿片受体激动剂U50488H(1.5 mg/kg)发现,U50488H+I/R组大鼠心肌梗死面积明显低于I/R组的大鼠心肌梗死面积(P0.01);血浆中CK和LDH的含量也明显降低(P0.01)。静脉给予选择性κ阿片受体拮抗剂Nor-BNI(1.5 mg/kg),Nor-BNI+U50488H+I/R组大鼠与I/R组大鼠心肌梗死范围无显著性差异(P0.05),血浆中CK和LDH的含量也无显著性差异(P0.05),但与U50488H+I/R组大鼠心肌梗死范围及血浆中CK和LDH相比,明显升高(P0.01)。 (4)各组以缺血30 min开始时为记录起始点,分别记录缺血0 min,缺血10 min,缺血30 min,再灌注30 min时间点的左心室收缩压(LVSP)及收缩功能(dp/dt_(max))和舒张功能(-dp/dt_(max)),发现IPC+I/R组在缺血10 min,缺血30 min,再灌注30 min时间点的LVSP和±dp/dt_(max)明显高于同时间点的I/R组(P0.05)。静脉给予κ阿片受体的特异性阻断剂Nor-BNI(2 mg/kg),Nor-BNI+IPC+I/R组在缺血10 min,缺血30 min,再灌注30 min时间点的LVSP和±dp/dt_(max)明显低于IPC+I/R组(P0.05)。 4、结论: (1)本研究首次表明,IPC可能促进机体内源性阿片肽Dyn的释放而增加了血浆Dyn水平,后者可通过激活κ阿片受体产生对心脏的保护作用。 (2)外源性阿片类物质U50488H激活κ阿片受体,能够显著降低I/R的心肌梗死面积,减少心肌细胞内蛋白酶的漏出,对I/R心肌具有直接保护作用。 (3)给予κ阿片受体的特异性阻断剂Nor-BNI,能够阻断IPC对于I/R心肌在收缩功能方面的改善,进一步证明κ阿片受体介导了IPC对I/R心肌的保护作用。
[Abstract]:Opioid and opioid receptors are widely distributed in the body, it has long been recognized that opioid peptide is a kind of central neuropeptides or neurotransmitters, only play a role in the central nervous system. In the past 20 years, the study found that the heart can also synthesize endogenous opioid peptides by autocrine or paracrine regulation of their function, its role is through the respective opioid receptor mediated by opioid receptors. Closely related with the heart function is mainly u, Delta and kappa three subtypes, which dominates in the cardiovascular system of opioid receptor subtype is kappa opioid receptor, dynorphin (Dyn) is an endogenous ligand of kappa opioid receptors.
The research confirmed that myocardial ischemic preconditioning (IPC) protects the heart against subsequent sustained myocardial ischemia reperfusion (I/R) injury tolerance improved, the protective effect of IPC on myocardial ischemia is mainly due to trigger the release of endogenous substances to achieve. Known in normal conditions, the heart can be synthesized and can activate Dyn kappa opioid receptor on the cardiovascular system regulation; in myocardial ischemia, endogenous Dyn may release increased and pathological process by activating kappa opioid receptors involved in myocardial ischemia. Therefore, this experiment was conducted to study whether the protective effect of endogenous Dyn in myocardial IPC plays an important role and whether the activation of kappa opioid receptor myocardial ischemia / reperfusion injury has a protective effect directly.
1, to:
(1) observed in the IPC process, the change of Dyn concentration in the plasma level of Dyn and the changes in the expression of mRNA in myocardium.
(2) with selective kappa opioid receptor agonist, to observe its effect on myocardial injury in I/R rats.
(3) with selective kappa opioid receptor antagonist, observed the effects of IPC on myocardial protection.
2, research methods:
(1) IPC model preparation methods: rat left anterior descending coronary artery (LAD) from the lower edge of the left atrial appendage about 2 mm thread ligation, LAD, myocardial ischemia 5 min, reperfusion 5 min, release the 3 cycle.
(2) I/R model preparation methods: ligation of LAD, myocardial ischemia 45 min, reperfusion 120 min. release
(3) determination of the physiological experimental technique was used to collect the blood of rats and cardiac function.
(4) to observe the changes of expression of Dyn in myocardium of mRNA rats by RT-PCR method.
(5) the plasma level of Dyn was measured by radioimmunoassay, creatine kinase (CK) and lactate dehydrogenase (LDH) level.
(6) the area of myocardial infarction was detected by double staining method.
3, the main results:
(1) blood collection rats, centrifuged supernatant plasma, plasma concentrations were measured in each group of rats Dyn, IPC group (ischemia 5 min, reperfusion 5 min, after 3 cycles of sampling) were significantly higher than sham group (not given the same time of ischemia and reperfusion, and other treatments were the same as group IPC) (P0.05); the concentration of IPC group was significantly higher than that of con group (without any treatment) (P0.05).
(2) the myocardium of rats, and observe the expression of Dyn mRNA in myocardial tissue of each group, IPC group (ischemia 5 min, reperfusion 5 min, after 3 cycles of sampling) dynorphin gene expression level and the same time sham group (not given ischemia and reperfusion, and other treatments were the same as group and con group (IPC) without any treatment) compared with no significant difference (P0.05).
(3) intravenous selective kappa opioid receptor agonist U50488H (1.5 mg/kg), the area of myocardial infarction in the rats of U50488H+I/R group was significantly lower than that in the area of myocardial infarction in I/R group (P0.01); the content of CK and LDH in plasma were significantly decreased (P0.01). Intravenous administration of a selective kappa opioid receptor antagonist Nor-BNI (1.5 mg/kg), there was no significant difference between the group Nor-BNI+U50488H+I/R rats and I/R rats myocardial infarction (P0.05), and there was no significant difference in the contents of CK and LDH in plasma (P0.05), but compared with the LDH and CK of myocardial infarct and plasma of rats in the U50488H+I/R group, was significantly increased (P0.01).
(4) each to 30 min ischemia at the start of recording starting point, were recorded 0 min ischemia, ischemia 10 min, 30 min ischemia, left ventricular systolic 30 min reperfusion time point pressure (LVSP) and systolic function (dp/dt_ (max)) and diastolic function (-dp/dt_ (max)), IPC+ in group I/R, 10 min ischemia, ischemia 30 min, reperfusion 30 min time point and LVSP + dp/dt_ (max) was significantly higher than that in group I/R at the same time point (P0.05). Intravenous opioid receptor specific antagonist Nor-BNI (2 mg/kg), group Nor-BNI+IPC+I/R in 10 min ischemia, ischemia 30 min, 30 min reperfusion time point and LVSP + dp/dt_ (max) was significantly lower than that of IPC+I/R group (P0.05).
4, conclusion:
(1) this is the first study to show that IPC may promote the endogenous opioid peptide Dyn release and increased plasma Dyn levels, which may be through the activation of kappa opioid receptor had a protective effect on the heart.
(2)澶栨簮鎬ч樋鐗囩被鐗╄川U50488H婵，

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